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The Influence of Downregulation of DEPTOR Expression by RNA Interference on the Proliferation and Apoptosis of Human Multiple Myeloma Cells in vitro |
ZHANG Hao-ran, ZENG Zhi-yong, CHEN Jun-min |
Department of Hematology, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China |
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Abstract Objective: To construct short hairpin RNA (shRNA) lentiviral vectors targeting human DEPTOR gene, detect its effect of gene silence and investigate the effect of DEPTOR on the proliferation and apoptosis in human multiple myeloma (MM) RPMI-8226 cells. Methods: Four human DEPTOR siRNA sequences were designed and then cloned into hU6-MCS-CMV-EGFP(GV115)-shRNA vector. The above recombinants were transfected into 293T cells by means of lipofectamine mediation. The gene silencing efficacy of these 4 siRNA sequences was compared in Western blot analysis. The GV115-shRNA was co-transfected into 293T cells. The most effective GV115-shRNA was screened out by Western blot. GV115-shRNA and lentiviral packaging plasmid were co-transfected into packging cell line 293T. Culture supernatants were harvested and concentrated to generate the lentivirus encoding DEPTOR-RNAi. The lentivirus particles were packaged and DEPTOR specific shRNA was transmitted into RPMI-8226 cells after screening for the valid shRNA. DEPTOR silencing efficiency was determined by Real-time PCR at mRNA level and Western blot at protein level. MTT method was used to detect the proliferation of the cells. Flow cytometry was used to detect the cell apoptosis. Changes of cleaved caspase-3 and cleaved PARP were analyzed by Western blot. Results: The constructed revealed shRNA plasmids were proved to be correct by PCR and sequencing. shRNA plasmid (GV115-shRNA2), which efficiently silenced DEPTOR, was screened out. GV115-shRNA2 and lentiviral packaging plasmid were successfully packaged with a titer of 1?109 TU/ml. EGFP expression could be detected in RPMI-8226 after infection of the recombinant lentivirus. The expression of DEPTOR decreased obviusly detected by Real-time PCR and Western blot. Down-regulation of DEPTOR expression distinctly decrease the proliferation rates of MM line (P<0.05), induce tumor cell apoptosis (P<0.01). DEPTOR shRNA could increase expression of cleaved caspase-3 and cleaved PARP and Bax. The expression of protein Bcl-2 was decreased, while the PI3K/Akt signaling pathway was blocked (P<0.01). Conclusion: The DEPTOR shRNA recombinant lentivirus vectors was successfully constructed, and the shRNA can significantly inhibit the expression level of DEPTOR in RPMI-8226 cells. DEPTOR shRNA will induce MM cell apoptosis and inhibit its proliferation. In addition, PI3K/Akt pathway may be involved in the process of apoptosis.
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Received: 17 December 2012
Published: 25 May 2013
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[1] Guertin D A, Sabatini D M. Defining the role of mTOR in cancer. Cancer Cell, 2007, 12(1): 9-22. [2] Sabatini D M. mTOR and cancer: insights into a complex relationship. Nat Rev Cancer, 2006, 6(9): 729-734. [3] Bentzinger C F, Romanino K, Clotta D, et al. Skeletal muscle-specific ablation of raptor, but not of rictor, causes metabolic changes and results in muscle dystrophy. Cell Metab, 2008, 8(5): 411-424. [4] Peterson T R, Laplante M, Thoreen C C, et al. DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell, 2009, 137: 873-886. [5] Efeyan A, Sabatini D M. mTOR and cancer: many loops in one pathway. Curr Opin Cell Biol, 2010, 22: 169-176. [6] Ghosh P, Wu M, Zhang H, et al. mTORC1 signaling requires proteasomal function and the involvement of CUL4-DDB1 ubiquitin E3 ligase. Cell Cycle, 2008, 7: 373-381. [7] Zhao Y, Xiong X, Sun Y. DEPTOR, an mTOR inhibitor, is a physiological substrate of SCF(βTrCP) E3 ubiquitin ligase and regulates survival and autophagy. Mol Cell, 2011, 44: 304-316. [8] Morgensztern D, Mcleod H L. PI3K/Akt/mTOR pathway as a target for cancer therapy. Anticancer Drugs, 2005, 16: 797-803. [9] McMillin D W, Ooi M, Delmore J, et al. Antimyeloma activity of the orally bioavailable dual phosphatidylinositol 3-kinase?mammalian target of rapamycin inhibitor NVP-BEZ235. Cancer Res, 2009, 69: 5835-5842. [10] Abbas-Terki T, Blanco-Bose W, Déglon N, et al. Lentiviral-mediated RNA interference. Hum Gene Ther, 2002, 13: 2197-2201. [11] Stewart S A, Dykxhoorn D M, Palliser D, et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA, 2003, 9: 493-501. [12] Strillacci A, Griffoni C, Spisni E, et al. RNA interference as a key to knockdown overexpressed cyclooxygenase-2 gene in tumour cells. Br J Cancer, 2006, 94: 1300-1310. [13] Charames G S, Bapat B. Cyclooxygenese-2 knockdown by RNA interference in colon cancer. Int J Oncol, 2006, 28: 543-549. [14] Kyle R A, Rajkumar S V. Multiple myeloma. N Engl J Med, 2004, 351: 1860-1873. [15] Kyle R A, Rajkumar S V. Multiple myeloma. Blood, 2008, 111: 2962-2721. [16] Elbashir S M, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001, 411: 494-498. [17] Cioca D P, Aoki Y, Kiyosawa K. RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines. Cancer Gene Ther, 2003, 10: 125-133. [18] Smith-Arica J R, Bartlett J S. Gene therapy: recombinant adeno-associated virus vectors. Curr Cardiol Rep, 2001, 3: 43-49. [19] Wiznerowicz M, Trono D. Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J Virol, 2003, 77: 8957-8961. [20] Rubinson D A, Dillon C P, Kwiatkowski A V, et al. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet, 2003, 33: 401-406. [21] Neschadim A, McCart J A, Keating A, et al. A roadmap to safe, efficient, and stable lentivirus-mediated gene therapy with hematopoietic cell transplantation. Biol Blood Marrow Transplant, 2007, 13: 1407-1416. [22] Zhang Y, Ni J, Zhou G, et al. Cloning, expression and characterization of the human NOB1 gene. Mol Biol Rep, 2005, 32: 185-189. [23] Wolf B B, Schuler M, Echeverri F, et al. Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation. J Biol Chem, 1999, 274: 30651-30656. [24] Zhao R J, Wang H, Wang G B, et al. Relationship of Caspase family and apoptosis. Chinese Journal of Animal Science, 2010, 17: 73-78. [25] Visconti R, D’Adamio L. Functional cloning of genes regulating apoptosis in neuronal cells. Methods Mol Biol, 2007, 399: 125-131. [26] Kuribayashi K, Mayes P A, El-Deiry W S. What are caspases 3 and 7 doing upstream of the mitochondria? Cancer Biol Ther, 2006, 5: 763-765. [27] Lockshin R A. Programmed cell death: history and future of a concept. J Soc Biol, 2005, 199: 169-173. [28] Mitsiades N, Mitsiades C S, Poulaki V, et al. Biologic sequelae of nuclear factor-kappaB blockade in multiple myeloma: therapeutic applications. Blood, 2002, 99(11): 4079-4086. [29] Sumantran V N, Lee D S, Baker V V, et al. A bcl-x(S) adenovirus demonstrates therapeutic efficacy in an ascites model of human breast cancer. J Soc Gynecol Investig, 2000, 7(3): 184-189. |
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